Wanyi Li

Expression of mouse beta-defensin-3 in MDCK cells and its anti-influenza-virus activity

Influenza (flu) pandemics have presented a threat to human health in the past century. Because of outbreaks of avian flu in humans in some developing countries in recent years, humans are more eager to find a way to control flu. Mammalian beta-defensins (β-defensins) are associated primarily with mucosal and skin innate immunity. Previous studies have demonstrated antimicrobial properties of a variety of defensin peptides. We have identified the presence of mouse β-defensin 1, 2, and 3 genes (Mbd-1, 2, and 3) in trachea and lung tissues by RT-PCR before and after infection with influenza virus. We constructed a eukaryotic expression plasmid containing Mbd-3, pcDNA 3.1(+)/MBD-3, and the plasmid was introduced into Madin–Darby canine kidney (MDCK) cells by transfection. The expression of Mbd-3 in MDCK cells was verified by immunofluorescence test, RT-PCR, and Western blot. The pcDNA 3.1(+)/MBD-3 plasmid was injected into mice to observe its effect against influenza A virus (IAV) in vivo. Mouse β-defensin genes could be expressed in trachea and lung tissues before IAV infection, but expression of Mbd-2 and Mbd-3 was increased significantly after IAV infection. The survival rate of mice with MBD-3 against IAV challenge was 71.43%, and MDCK cells with MBD-3 could clearly inhibit IAV replication. The results demonstrated that mouse β-defensins possess anti-influenza virus activity, suggesting that mouse β-defensins might be used as agents to prevent and treat influenza.

Antiviral activity of recombinant mouse β-defensin 3 against influenza A virus in vitro and in vivo.

Background: Influenza causes significant morbidity and mortality. Mammalian β-defensins are small peptides of about 4.5–6 kDa in mass and are effectors of the innate immune response with potent antimicrobial activity. In this paper, we focused on the anti-influenza A activity of the recombinant mouse β-defensin 3 (rMBD-3) in vivo and in vitro. Methods: The rMBD-3 peptide was added to Madin-Darby canine kidney (MDCK) cells at different stages of influenza A virus (IAV) A/PR/8/34 (H1N1) infection and its virus inhibitory properties were determined. Mice were infected with IAV and treated with rMBD-3 peptide from 12 h post-infection. The effect of rMBD-3 peptide was determined by pulmonary viral load, pathology and mortality. In addition, the expression of interleukin (IL)-12, interferon (IFN)-γ and tumour necrosis factor (TNF)-α genes in mice with or without rMBD-3 treatment was determined by semi-quantitative reverse transcriptase PCR. Results: rMBD-3 was shown to protect MDCK cells against IAV infection and had a major role in inhibition of adsorption and uptake by cells infected with IAV. Following the addition of 100 μg/ml rMBD-3 to MDCK cells medium, approximately 80% of cells were protected from infection in vitro. rMBD-3 given by tail vein injection (10 mg/kg/day) was the most effective method to improve the survival rate of the mice. Treatment with rMBD-3 was found to up-regulate IFN-γ and IL-12 gene expression, but reduced expression of the TNF-α gene. Conclusions: These results demonstrate that rMBD-3 possesses anti-influenza virus activity both in vivo and in vitro that might be of therapeutic use.

Immunogenicity of multiple-epitope antigen gene of HCV carried by novel biodegradable polymers.

In order to develop a promising vaccine candidate utilizing a combined approach to induce both antibody production and T-cell activity, the DNA fragment containing MA of HCV with five conserved epitopes was synthesized. Two types of HCV vaccine candidates (the DNA type and DNA/polymers) were constructed using MA. PLA-PEG-PLA and PLGA-PEG-PLGA were synthesized and used as micelles with encapsulated plasmid pcDNA3.1(+)-MA. The preparation of copolymers, the cloning and analysis of recombinant plasmid DNA, in vitro expression, and immunogenicity in transgenic mice were evaluated in detail. The results indicated that even single immunization and oral immunization with DNA/polymers achieved satisfying immune responses in vivo tests. As biodegradable and nontoxic triblock copolymers, the novel copolymers demonstrated a great advantage, as they made long-term and single-immunizing vaccines possible; in addition, the copolymers showed a better adjuvant effect and scarcely any side effects.

Association of TNF-α Gene Promoter Polymorphisms With Susceptibility of Cervical Cancer in Southwest China

Objective: Variations in the tumor necrosis factor-alpha (TNF-α) gene may lead to changes in the level of TNF-α associated with the susceptibility to cancer. This study is designed to determine the association of TNF-α promoter polymorphisms with the susceptibility of cervical cancer among women living in Southwest China. Methods: Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing, we analyzed the genotype and allele distributions of 4 single-nucleotide polymorphisms (SNP) of TNF-α gene in 239 cervical cancer patients and 110 controls. Results: Compared to controls, cervical cancer patients show a significant increase in the frequency of GA genotype at −308 G/A, and a significantly decreased frequency of the CT genotype. A significant decrease in the frequency of the T allele at −857 C/T ( P <0.05) was also found. No significant differences of SNP genotype and allele at TNF-α-863 C/A and −238 G/A were observed between the 2 groups. Conclusion: An SNP at TNF-α −857 C/T and −308 G/A, but not −863 C/A or −238 G/A, were significantly associated with an increased risk of cervical cancer in the studied population. * TNF-α : tumor necrosis factor-alpha PCR-RFLP : polymerase chain reaction-restriction fragment length polymorphism SNP : single-nucleotide polymorphism HPV : human papillomavirus EGF : epidermal growth factor CIN : cervical intraepithelial neoplasia EDTA : ethylenediaminetetraacetic acid OR : odds ratios CI : confidence interval ICC : invasive cervical cancer

Immune effects against influenza A virus and a novel DNA vaccine with co-expression of haemagglutinin- and neuraminidase-encoding genes.

The high variability of influenza virus causes difficulties in the control and prevention of influenza, thus seeking a promising approach for dealing with these problems is a hot topic. Haemagglutinin (HA) and neuraminidase (NA) are major surface antigens of the influenza virus, and provide effective protection against lethal challenges with this virus. We constructed a DNA vaccine (pHA-IRES2-NA) that co-expressed both HA and NA, and compared its protective efficacy and immunogenic ability with that of singly expressed HA or NA, or a mixture of the two singly expressed proteins. Our findings showed that both HA and NA proteins expressed by pHA-IRES2-NA could be detected in vivo and in vitro. The protection of DNA vaccines was evaluated by serum antibody titres, residual lung virus titres and survival rates of the mice. In the murine model, immunization of pHA-IRES2-NA generated significant anti-HA and anti-NA antibody, increased the percentage of CD8(+) cells and gamma interferon-producing CD8(+) cells and the ratio of Th1/Th2 (T helper) cells, which was comparable to the effects of immunization with HA or NA DNA alone or with a mixture of HA and NA DNA. All the mice inoculated by pHA-IRES2-NA resisted the lethal challenge by homologous influenza virus and survived with low lung virus titre. In addition, previous studies reported that co-expression allowed higher-frequency transduction compared to co-transduction of separated vector systems encoding different genes. The novel HA and NA co-expression DNA vaccine is a successful alternative to using a mixture of purified HA and NA proteins or HA and NA DNA.

Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus

Influenza (flu) pandemics have exhibited a great threat to human health throughout history. With the emergence of drug-resistant strains of influenza A virus (IAV), it is necessary to look for new agents for treatment and transmission prevention of the flu. Defensins are small (2–6 kDa) cationic peptides known for their broad-spectrum antimicrobial activity. Beta-defensins (β-defensins) are mainly produced by barrier epithelial cells and play an important role in attacking microbe invasion by epithelium. In this study, we focused on the anti-influenza A virus activity of mouse β-defensin 1 (mBD1) and β defensin-3 (mBD3) by synthesizing their fusion peptide with standard recombinant methods. The eukaryotic expression vectors pcDNA3.1(+)/mBD1-mBD3 were constructed successfully by overlap-PCR and transfected into Madin-Darby canine kidney (MDCK) cells. The MDCK cells transfected by pcDNA3.1(+)/mBD1-mBD3 were obtained by G418 screening, and the mBD1-mBD3 stable expression pattern was confirmed in MDCK cells by RT-PCR and immunofluorescence assay. The acquired stable transfected MDCK cells were infected with IAV (A/PR/8/34, H1N1, 0.1 MOI) subsequently and the virus titers in cell culture supernatants were analyzed by TCID50 72 h later. The TCID50 titer of the experimental group was clearly lower than that of the control group (p < 0.001). Furthermore, BALB/C mice were injected with liposome-encapsulated pcDNA3.1(+)/mBD1-mBD3 through muscle and then challenged with the A/PR/8/34 virus. Results showed the survival rate of 100% and lung index inhibitory rate of 32.6% in pcDNA3.1(+)/mBD1-mBD3group; the TCID50 titer of lung homogenates was clearly lower than that of the control group (p < 0.001). This study demonstrates that mBD1-mBD3 expressed by the recombinant plasmid pcDNA3.1(+)/mBD1-mBD3 could inhibit influenza A virus replication both in vitro and in vivo. These observations suggested that the recombinant mBD1-mBD3 might be developed into an agent for influenza prevention and treatment.

Relationship between the iceA gene of Helicobacter pylori and clinical outcomes

Background The complex pathogenesis of Helicobacter pylori (H. pylori) and the features of the host influence the diverse clinical outcomes. A mass of studies about virulence genes have accelerated the exploration of pathogenesis of H. pylori infection. Induced by contact with epithelium gene A (iceA) is one of the biggest concerned virulence genes. In this study, we explored the relationship between iceA and the magnitude of the risk for clinical outcomes and the prevalence of iceA-positive H. pylori in People’s Republic of China and other countries. Methods We searched the electronic databases of PubMed, Embase, CNKI, VIP, and Wanfang by literature search strategy. The studies conforming to the inclusion criteria were assessed. With these data, we systematically analyzed the relationship between the iceA gene of H. pylori and clinical outcomes. Results Nineteen articles with 22 studies, a total of 2,657 cases, were involved in the study. The iceA1 gene was significantly associated with peptic ulcer disease (odds ratio =1.28, 95% confidence interval =1.03–1.60; P=0.03), especially in People’s Republic of China (odds ratio =1.40, 95% confidence interval =1.07–1.83; P=0.01). Moreover, the prevalence of iceA1 was significantly higher than iceA2 in People’s Republic of China (P<0.0001). The prevalence of both iceA1 and iceA2 was significantly different (P<0.0001) in People’s Republic of China and in other countries. Conclusion The system analysis showed that infection with the iceA1-positive H. pylori significantly increased the overall risk for peptic ulcer disease, especially in People’s Republic of China. The iceA2 gene status and clinical outcome of H. pylori infection have no significant correlation. H. pylori iceA1 genotype is the major epidemic strain in People’s Republic of China.

Preparation and characterization of a new monoclonal antibody against CXCR4 using lentivirus vector

CXCR4 is a member of chemokine receptors and plays a vital role in numerous diseases and cancer processes, which makes the CXCR4/CXCL12 chemotactic axis a potential therapeutic target. In this study, we used lentiviral vectors as a novel technology to produce a monoclonal antibody against CXCR4. Lentivirus vector pLV-CXCR4-Puro was successfully constructed and a hybridoma cell line 1A4 was generated. The CXCR4 monoclonal antibody (MAb) 1A4 had high titer and affinity, and the isotype was identified as IgG1a. The recombinant lentivirus vector could effectively stimulate the production of 39kDa CXCR4 antibody in vivo after immunization. Western blot analysis showed that the MAb could recognize the CXCR4 antigen expressed on transfected 293T cells as well as various human cancer cell lines. Immunofluorescence assays showed that MAb 1A4 mainly localized and strongly stained on the membrane of transfected 293T cells. Immunohistochemistry assays demonstrated that 1A4 could recognize strong expression of CXCR4 on the hepatocellular carcinoma (HCC). Thus, the method using lentiviral vectors may have application on effective and large-scale production of the CXCR4 monoclonal antibody, which will be a potential tool for the diagnosis and treatment of human cancers.

Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34oC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

Digital gene expression analysis in mice lung with coinfection of influenza and streptococcus pneumoniae

Influenza A virus (IAV) and Streptococcus pneumoniae (SP) are two major upper respiratory tract pathogens that can also cause infection in polarized bronchial epithelial cells to exacerbate disease in coinfected individuals which may result in significant morbidity. However, the underlying molecular mechanism is poorly understood. Here, we employed BALB/c ByJ mice inflected with SP, IAV, IAV followed by SP (IAV+SP) and PBS (Control) as models to survey the global gene expression using digital gene expression (DGE) profiling. We attempt to gain insights into the underlying genetic basis of this synergy at the expression level. Gene expression profiles were obtain using the Illimina/Hisseq sequencing technique, and further analyzed by enrichment analysis of Gene Ontology (GO) and Pathway function. The hematoxylin-eosin (HE) staining revealed different tissue changes in groups during which IAV+SP group showed the most severe cell apoptosis. Compared with Control, a total of 2731, 3221 and 3946 differentially expressed genes (DEGs) were detected in SP, IAV and IAV+SP respectively. Besides, sixty-two GO terms were identified by Gene Ontology functional enrichment analysis, such as cell killing, biological regulation, response to stimulus, signaling, biological adhesion, enzyme regulator activity, receptor regulator activity and translation regulator activity. Pathway significant enrichment analysis indicated the dysregulation of multiple pathways, including apoptosis pathway. Among these, five selected genes were further verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). This study shows that infection with SP, IAV or IAV+SP induces apoptosis with different degrees which might provide insights into the molecular mechanisms to facilitate further research.